Methods and compositions for treating equine conditions using recombinant self-complementary adeno-associated virus

ABSTRACT

Methods and compositions for treating symptoms of conditions such as but not limited to osteoarthritis in horses. The methods may feature direct intraarticular injection of a recombinant self-complementary adeno-associated virus (sc-rAAV) with a vector adapted to express a modified IL-1 Ra peptide. The methods of the present invention may express a therapeutically effective amount of the modified IL-1 Ra peptide so as to ameliorating symptoms associated with the condition being treated.

CROSS REFERENCE

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/377,281 filed Aug. 19, 2016, the specification(s) of which is/areincorporated herein in their entirety by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. AR054903awarded by National Institutes of Health. The government has certainrights in the invention.

REFERENCE TO SEQUENCE LISTING

Applicant asserts that the information recorded in the form of an AnnexC/ST.25 text file submitted under Rule 13ter.1(a), entitledCLAIM_16_01_PCT_Sequence_Listing_ST25.txt, is identical to that formingpart of the international application as filed. The content of thesequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to gene therapy and compositions for genetherapy, more particularly to recombinant self-complementaryadeno-associated virus (sc-rAAV) and methods of treating conditions orsymptoms of conditions using sc-rAAV.

BACKGROUND OF THE INVENTION

IL-1 is a powerful mediator of both chondrocytic chondrolysis andsuppression of matrix synthesis by chondrocytes. Together, these twoprocesses are highly destructive to cartilage. IL-1 has also been shownto inhibit chondrogenesis but at the same time promote certain aspectsof the osteogenic differentiation that could help account for theformation of osteophytes and sclerosis of sub-chondral bone. In studyingcartilage recovered from human joints with OA, the production of IL-1 bychondrocytes was found to be highly elevated and sustained in anautocrine fashion. Moreover, the cells did not produce IL-1Ra. Thissuggests enhanced autocrine and paracrine activation of chondrocytes byIL-1 in the absence of its major physiological inhibitor during OA.Enhanced responsiveness of chondrocytes to IL-1 in OA was also indicatedby increased expression of the type I IL-1 receptor, the signalingreceptor, on OA chondrocytes. The local production and consumption ofIL-1 by chondrocytes may help explain why concentrations of IL-1 insynovial fluid tend to be low, even in OA. Also, genetic analyses haveidentified single nucleotide polymorphisms (SNPs) in the human geneencoding IL-1Ra (IL1RN) and regulatory elements that correlate with theincidence and severity of certain types of OA.

Targeted drug delivery is a major problem for the intra-articulartreatment of joint diseases. Molecules of all sizes, as well asparticles, are rapidly removed from joints via the lymphatics,subsynovial capillaries, or both. This makes it difficult to achievesustained, therapeutic doses of anti-OA drugs in joints. To addressthis, small molecules can be delivered systemically, but proteins aredifficult to deliver in this fashion because of size-dependentconstraints in crossing the fenestrated endothelium of the synovialcapillaries. Moreover, systemic delivery exposes non-target sites tohigh doses of the therapeutic, leading to unwanted side-effects. Therapid egress of proteins from joints, with half-lives typically of a fewhours, makes intra-articular delivery potentially ineffective. As anexample, recombinant IL-1Ra (Kineret, Amgen Biologicals) is delivered bydaily subcutaneous injection in effort to treat symptoms of RA. However,daily delivery fails to maintain therapeutic serum levels of IL-1Rabetween injections (Evans et al., 1996, Human Gene Therapy, 7:1261-1290;Evans et al., 2005, PNAS 102 (24): 8698-8703). Some studies have used exvivo gene transfer for introducing IL-1Ra to treat OA. However, theseapproaches are laborious and have not seemed to provide long-term geneexpression (Frisbie et al., 2002, Gene Therapy 9(1): 12-20). Also,several studies describe the use of a dual variabledomain-immunoglobulin (DVD-Ig) targeting IL-1alpha and IL-1beta (e.g.,ABT-981) for treating osteoarthritis (Kamath et al., 2011,Osteoarthritis and Cartilage 19S1:S64; Wang et al., 2015, Osteoarthritisand Cartilage 23:A398-399; Wang et al., 2014, Osteoarthritis andcartilage 22:S462-S463; Lacy et al., 2015, mAbs 7(3):605-619; Wu et al.,2009, mAbs 1(4):339-347; Wang et al., 2014, Scientific Abstracts SAT0448pg. 756; Goss et al., 2014, Scientific Abstracts SAT0447 pg. 755-756; US2015/0050238; Wang et al., 2014 ACR/ARHP Annual Meeting Abstract Number2237; Wang et al., 2015 ACR/ARHP Annual Meeting Abstract Number 318).However, these peptides require repeated systemic introduction (e.g., 4doses every 2 weeks or 3 doses every 4 weeks, e.g., by subcutaneousinjection or intravenous infusion) because of the relatively shorthalf-life (Wang et al., 2015, Osteoarthritis and Cartilage 23:A398-399;Wang et al., 2014, Osteoarthritis and cartilage 22:S462-S463; Evans etal., 2005, PNAS 102 (24): 8698-8703).

The present invention features methods and compositions for delivering atherapeutic gene product (e.g., IL-1Ra) in a sustained manner to alocation of interest, e.g., joints, in horses. The present inventionalso features methods and compositions for treating symptoms ofconditions such as but not limited to osteoarthritis. The presentinvention also features methods and compositions for providing a horse atherapeutically effective amount of a therapeutic gene product (e.g.,IL-1Ra). The methods and compositions may feature a recombinantself-complementary adeno-associated virus (sc-rAAV), wherein the sc-rAAVcomprises an engineered capsid and a vector (e.g., a sc-rAAV vector)packaged within the capsid. The vector may comprise a transgene (e.g., anucleotide sequence encoding a protein of interest, e.g., a therapeuticgene product, e.g., IL-1Ra or a modified version thereof) operablylinked to a promoter (e.g., a constitutive promoter). The therapeuticgene product may be delivered to a location of interest, e.g., a joint.For example, for treating osteoarthritis, the sc-rAAV may be introducedinto cells (e.g., chondrocytes, synoviocytes, etc.) in a joint viadirect intraarticular injection. The present invention is not limited tothe aforementioned conditions, nor the location of interest (e.g.,joint).

It is noted that Goodrich et al. (Molecular Therapy-Nucleic Acids, 2013,2:e70) generally discloses a method of treating osteoarthritis usingscAAV-delivered IL-1Ra. However, Goodrich et al. does not specificallyidentify or enable any particular IL-1Ra sequence, e.g., an IL-1Rasequence according to the present invention. In particular, the field ofgene therapy is an unpredictable area wherein one cannot assume that anyparticular gene sequence for a protein of interest will be efficientlyexpressed.

SUMMARY OF THE INVENTION

The present invention features a recombinant self-complementaryadeno-associated virus (sc-rAAV). In some embodiments, the sc-rAAVcomprises an engineered AAV capsid and a vector packaged within thecapsid, wherein the vector comprises a modified IL-1Ra gene operablylinked to a promoter and the modified IL-1Ra gene is at least 95%identical to SEQ ID NO: 2. In some embodiments, the promoter comprises aCMV promoter. In some embodiments, the engineered capsid comprises atleast a portion of serotype AAV2 and at least a portion of serotypeAAV6. In some embodiments, the engineered capsid comprises at least aportion of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10, AAV11, or a combination thereof. In some embodiments, thevector further comprises SV40 and bovine growth hormone (bGH)polyadenylation sequences. In some embodiments, the vector furthercomprises SV40 splice donor (SD) and splice acceptor (SA) sites. In someembodiments, the vector comprises sc-rAAV2.5Hu-IL-1Ra. In someembodiments, the sc-rAAV is part of a composition.

In some embodiments, the sc-rAAV comprises an engineered AAV capsid anda vector packaged within the capsid, wherein the vector comprises amodified IL-1Ra gene operably linked to a promoter and the modifiedIL-1Ra gene encodes IL-1Ra protein according to SEQ ID NO: 6 or SEQ IDNO: 7.

The present invention features a method of providing a horse in needthereof (e.g., a horse diagnosed with or at risk for osteoarthritis) atherapeutically effective amount of interleukin-1 receptor agonist(IL-1Ra) peptide. In some embodiments, the method comprises introducinginto a location of interest (e.g., via intraarticular injection) acomposition comprising a recombinant self-complementary adeno-associatedvirus (sc-rAAV) according to the present invention. The sc-rAAVtransduces the vector into cells in the location of interest, whereinthe modified IL-1Ra gene is expressed so as to provide the horse withthe therapeutically effective amount of said IL-1Ra peptide.

The present invention also features a method of ameliorating symptoms ofosteoarthritis in a horse. In some embodiments, the method comprisesintroducing into a location of interest (e.g., via direct intraarticularinjection) a composition comprising a recombinant self-complementaryadeno-associated virus (sc-rAAV) according to the present invention. Thesc-rAAV transduces the vector into cells in the location of interest,wherein the modified IL-1Ra gene is expressed so as to provide the horsewith an amount of IL-1Ra peptide effective for ameliorating symptomsassociated with osteoarthritis.

The present invention also features a method of repairing cartilage in ahorse in need thereof (e.g., a horse diagnosed with or at risk fordeveloping osteoarthritis). In some embodiments, the method comprisesintroducing into a location of cartilage (e.g., via directintraarticular injection) a composition comprising a recombinantself-complementary adeno-associated virus (sc-rAAV) according to thepresent invention. The sc-rAAV transduces the vector into cells in thelocation of cartilage, wherein the modified IL-1Ra gene is expressed soas to provide the horse with IL-1Ra peptide effective for repairingcartilage.

The present invention also features a method of providing interleukin-1receptor agonist (IL-1Ra) peptide to an area of inflammation. In someembodiments, the method comprises introducing into a location ofinflammation (e.g., via intraarticular injection) a compositioncomprising a recombinant self-complementary adeno-associated virus(sc-rAAV) according to the present invention. The sc-rAAV transduces thevector into cells in the location of inflammation, wherein the modifiedIL-1Ra gene is expressed so as to provide the cells in the location ofinflammation a therapeutically effective amount of IL-1Ra peptideeffective for reducing inflammation.

In some embodiments, the location of interest is a joint, synovium,subsynovium, joint capsule, tendon, ligament, cartilage, orperi-articular muscle of the horse. In some embodiments, the cells arechondrocytes, synoviocytes, or a combination thereof.

In some embodiments, the method is performed a second time at a timepoint after a time when the method is performed first. In someembodiments, the time point is at least 3 months. In some embodiments,the method further comprises co-introducing a secondary therapy (e.g., aglucocorticoid, hyaluronan, platelet-rich plasma, recombinant, horseIL-1Ra, or a combination thereof) to the location of interest incombination with the composition.

The present invention also features a method of delivering IL-1Rapeptide to a chondrocyte or synoviocyte. In some embodiments, the methodcomprises contacting the chondrocyte or synoviocyte with a recombinantself-complementary adeno-associated virus (sc-rAAV) according to thepresent invention, e.g., an engineered adeno-associated virus (AAV)capsid comprising at least a portion of serotype 2 and at least aportion of serotype 6 and a vector packaged within the capsid, whereinthe vector comprises a modified IL-1Ra gene operably linked to a CMVpromoter and the modified IL-1Ra gene is at least 95% identical to SEQID NO: 2. The sc-rAAV transduces the vector into the chondrocyte orsynoviocyte and the modified IL-1Ra gene is expressed to as to provideIL-1Ra peptide to the chondrocyte or synoviocyte.

For the aforementioned methods and compositions (e.g., a method ofproviding a horse in need thereof a therapeutically effective amount ofinterleukin-1 receptor agonist (IL-1Ra) peptide, a method ofameliorating symptoms of osteoarthritis or rheumatoid arthritis in ahorse, a method of delivering IL-1Ra peptide to a chondrocyte orsynoviocyte, a composition comprising a recombinant self-complementaryadeno-associated virus (sc-rAAV), a recombinant self-complementaryadeno-associated virus (sc-rAAV) vector comprising a modified IL-1Ragene operably linked to a CMV promoter, a method of repairing cartilagein a canine in need thereof, a method of providing interleukin-1receptor agonist (IL-1Ra) peptide to an area of inflammation, etc.), themodified IL-1Ra gene may be at least 95% identical SEQ ID NO: 2 andencode IL-1Ra according to SEQ ID NO: 6 or SEQ ID NO: 7.

Any feature or combination of features described herein are includedwithin the scope of the present invention provided that the featuresincluded in any such combination are not mutually inconsistent as willbe apparent from the context, this specification, and the knowledge ofone of ordinary skill in the art. Additional advantages and aspects ofthe present invention are apparent in the following detailed descriptionand claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Plasmid p-trsKS-cmv-opt-eq-il1-ra, which contains amodified cDNA encoding IL-1Ra protein under control of the CMV promoter.A modified sequence for the equine IL-1ra gene was removed from anon-expression vector using Notl and Agel restriction enzyme sites (NEB,Ipswich, Mass.), and ligated into a pTRs-ks mammalian expression vectorobtained from the UNC Vector Core (Chapel Hill, N.C.) containing a CMVpromoter. Ligations were performed using T4 ligase as per manufacturer'sinstructions (NEB). Constructs were transformed into DH10Electrocompetent cells (Invitrogen) and evaluated for ITR sites usingSmal enzyme sites (NEB). Once these sites were confirmed, the constructswere then transformed into SURE cells (Invitrogen), and evaluated againfor ITR sites. Constructs are ampicillin resistant. The total size ofthe construct is 5.754 kb in length.

TERMS

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which a disclosed invention belongs. The singularterms “a,” “an,” and “the” include plural referents unless contextclearly indicates otherwise. Similarly, the word “or” is intended toinclude “and” unless the context clearly indicates otherwise.“Comprising” means “including.” Hence “comprising A or B” means“including A” or “including B” or “including A and B.”

Suitable methods and materials for the practice and/or testing ofembodiments of the disclosure are described below. Such methods andmaterials are illustrative only and are not intended to be limiting.Other methods and materials similar or equivalent to those describedherein can be used. For example, conventional methods well known in theart to which the disclosure pertains are described in various generaland more specific references, including, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory Press, 1989; Sambrook et al., Molecular Cloning: A LaboratoryManual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates, 1992 (andSupplements to 2000); Ausubel et al., Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlowand Lane, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, 1999, the disclosures of which are incorporated intheir entirety herein by reference.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Although methods and materials similar or equivalent to those describedherein can be used to practice or test the disclosed technology,suitable methods and materials are described below. The materials,methods, and examples are illustrative only and not intended to belimiting.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Adeno-Associated Virus (AAV), Recombinant AAV (rAAV), and RecombinantSelf-Complementary AAV (Sc-rAAV):

AAV is a small virus (20 nm) in the family Parvoviridae. AAV is notknown to cause disease. AAV has recently been used to gene therapy for avariety of reasons including that it has been shown to have lowimmunogenicity, the ability to effectively transduce non-dividing cells,and the ability to infect a variety of cell and tissue types.Recombinant AAV (rAAV) does not contain native viral coding sequences.Recombinant AAV DNA is packaged into the viral capsid as a singlestranded molecule about 4600 nucleotides in length. Following infectionof the cell by the virus, the molecular machinery of the cell convertsthe single DNA strand into a double-stranded form. Only the doublestranded DNA form is useful to the proteins of the cell that transcribethe contained gene or genes into RNA. Self-complementary AAV (sc-rAAV)is an engineered form of rAAV that can form an intra-molecular doublestranded DNA template. Thus, upon infection, the two complementaryhalves of sc-rAAV will associate to form one double stranded DNA unitthat is ready for immediate replication and synthesis.

Expression:

The translation of a nucleic acid sequence into a protein. Proteins maybe expressed and remain intracellular, become a component of the cellsurface membrane, or be secreted into the extracellular matrix ormedium.

Operably Linked:

A first nucleic acid sequence is operably linked with a second nucleicacid sequence when the first nucleic acid sequence is placed in afunctional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.

Pharmaceutically Acceptable Vehicles:

Pharmaceutically acceptable carriers (vehicles), e.g., solutions, may beconventional but are not limited to conventional vehicles. For example,E. W. Martin, Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa., 15th Edition (1975) and D. B. Troy, ed. Remington: TheScience and Practice of Pharmacy, Lippincott Williams & Wilkins,Baltimore Md. and Philadelphia, Pa., 21^(st) Edition (2006) describecompositions and formulations suitable for pharmaceutical delivery ofone or more therapeutic compounds or molecules. In general, the natureof the carrier will depend on the particular mode of administrationbeing employed. In addition to biologically-neutral carriers,pharmaceutical compositions administered may contain minor amounts ofnon-toxic auxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Preventing, Treating, Managing, or Ameliorating a Condition:

“Preventing” a disease may refer to inhibiting the full development of acondition. “Treating” may refer to a therapeutic intervention thatameliorates a sign or symptom of a disease or pathological conditionafter it has begun to develop. “Managing” may refer to a therapeuticintervention that does not allow the signs or symptoms of a disease orcondition to worsen. “Ameliorating” may refer to the reduction in thenumber or severity of signs or symptoms of a disease or condition.

Sequence Identity:

The identity (or similarity) between two or more nucleic acid sequencesis expressed in terms of the identity or similarity between thesequences. Sequence identity can be measured in terms of percentageidentity; the higher the percentage, the more identical the sequencesare. Sequence similarity can be measured in terms of percentagesimilarity (which takes into account conservative amino acidsubstitutions); the higher the percentage, the more similar thesequences are. Methods of alignment of sequences for comparison are wellknown in the art. Various programs and alignment algorithms aredescribed in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman& Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl.Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988;Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res.16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8,155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994.Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailedconsideration of sequence alignment methods and homology calculations.The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biotechnology (NCBI, National Libraryof Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on theInternet, for use in connection with the sequence analysis programsblastp, blastn, blastx, tblastn and tblastx. Additional information canbe found at the NCBI web site. BLASTN may be used to compare nucleicacid sequences, while BLASTP may be used to compare amino acidsequences. If the two compared sequences share homology, then thedesignated output file will present those regions of homology as alignedsequences. If the two compared sequences do not share homology, then thedesignated output file will not present aligned sequences. TheBLAST-like alignment tool (BLAT) may also be used to compare nucleicacid sequences (Kent, Genome Res. 12:656-664, 2002). BLAT is availablefrom several sources, including Kent Informatics (Santa Cruz, Calif.)and on the Internet (genome.ucsc.edu). Once aligned, the number ofmatches is determined by counting the number of positions where anidentical nucleotide or amino acid residue is presented in bothsequences. The percent sequence identity is determined by dividing thenumber of matches either by the length of the sequence set forth in theidentified sequence, or by an articulated length (such as 100consecutive nucleotides or amino acid residues from a sequence set forthin an identified sequence), followed by multiplying the resulting valueby 100. For example, a nucleic acid sequence that has 1166 matches whenaligned with a test sequence having 1554 nucleotides is 75.0 percentidentical to the test sequence (1166÷1554*100=75.0). The percentsequence identity value is rounded to the nearest tenth.

Therapeutically Effective Amount:

A quantity of a specified agent sufficient to achieve a desired effectin a subject being treated with that agent. Such agents may includeIL-1Ra. For example, a therapeutically effective amount of IL-1Ra may bean amount sufficient to prevent, treat, or ameliorate symptoms ofosteoarthritis. The therapeutically effective amount of an agent usefulfor preventing, ameliorating, and/or treating a subject will bedependent on the subject being treated, the type and severity of theaffliction, and the manner of administration of the therapeuticcomposition.

Transduced:

A transduced cell is a cell into which a nucleic acid molecule has beenintroduced by molecular biology techniques. As used herein, the termtransduction encompasses all techniques by which a nucleic acid moleculemight be introduced into such a cell, including transfection withviruses or viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration. Such cells are sometimes called transformed cells.

Vector:

A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may lack the nucleic acid sequences that permitit to replicate in a host cell. A vector may also include a gene ofinterest, one or more selectable marker genes, other genetic elementsknown in the art, or any other appropriate insert.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features methods and compositions for delivering atherapeutic gene product (e.g., IL-1Ra) in a sustained manner to alocation of interest, e.g., a joint. The present invention also featuresmethods and compositions for treating symptoms of conditions such as butnot limited to osteoarthritis. The present invention also featuresmethods and compositions for providing an individual (e.g., a horse) atherapeutically effective amount of a therapeutic gene product (e.g.,IL-1Ra). The methods and compositions may feature a recombinantself-complementary adeno-associated virus (sc-rAAV), wherein the sc-rAAVcomprises an engineered capsid and a vector (an sc-rAAV vector) packagedwithin the capsid. The vector may comprise a transgene (e.g., anucleotide sequence encoding a protein of interest, e.g., a therapeuticgene product, e.g., IL-1Ra or a modified version thereof) operablylinked to a promoter (e.g., a constitutive promoter).

As previously discussed, the present invention features compositionscomprising a recombinant self-complementary adeno-associated virus(sc-rAAVs) vector. A non-limiting example of a sc-rAAV vector is shownin SEQ ID NO: 1 of Table 1 below. The sc-rAAV vector of SEQ ID NO: 1comprises a modified IL-1Ra gene (the sequence within SEQ ID NO: 1 thatencodes IL-1Ra is underlined). The sc-rAAV vector is not limited to SEQID NO: 1. In some embodiments, the sc-rAAV vector comprises a nucleicacid sequence for IL-1Ra according to SEQ ID NO: 2. The presentinvention is not limited to SEQ ID NO: 2.

The sc-rAAV vectors comprise a nucleic acid that encodes a peptide ofinterest. In some embodiments, the nucleic acid is at least 90%identical to SEQ ID NO: 2. In some embodiments, the nucleic acid is atleast 92% identical to SEQ ID NO: 2. In some embodiments, the nucleicacid is at least 94% identical to SEQ ID NO: 2. In some embodiments, thenucleic acid is at least 95% identical to SEQ ID NO: 2. In someembodiments, the nucleic acid is at least 96% identical to SEQ ID NO: 2.In some embodiments, the nucleic acid is at least 97% identical to SEQID NO: 2. In some embodiments, the nucleic acid is at least 98%identical to SEQ ID NO: 2. In some embodiments, the nucleic acid is atleast 99% identical to SEQ ID NO: 2. Non-limiting examples of suchnucleic acid sequences can be found in Table 1 below. For example, SEQID NO: 3 is a sequence for a modified IL-1Ra that is about 98% identicalto SEQ ID NO: 2; SEQ ID NO: 4 is a sequence for a modified IL-1Ra thatis about 99% identical to SEQ ID NO: 2; and SEQ ID NO: 5 is a sequencefor a modified IL-1Ra that is about 95% identical to SEQ ID NO: 2 (notethat the bold letters in Table 1 are nucleotide substitutions ascompared to SEQ ID NO: 2, and the codon underlined).

TABLE 1 SEQ ID NO: DESCRIPTION SEQUENCE 1 Equine Modified IL-1ra p-ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgttrs-KS Expression Vectorcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcaga Sequence with CMVgagggagtggggttcggtacccgttacataacttacggtaaatggccc promotor & ampicillingcctggctgaccgcccaacgacccccgcccattgacgtcaataatgac resistancegtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccggactctagaggatccggtactcgaggaactgaaaaaccagaaagttaactggtaagtttagtctttttgtcttttatttcaggtcccggatccggtggtggtgcaaatcaaagaactgctcctcagtggatgttgcctttacttctaggcctgtacggaagtgttacttctgctctaaaagctgcggaattgtacccgcggcccgggatccggtaccggtgccacc atggagatcagacgcagaagcgtgcgccacctgatcagcctgctgctgttcctgctgtacagcgagaccgcctgccaccccctgggcaagaggccctgcaagatgcaggccttcagaatctgggacgtgaatcagaaaaccttctacatgcgcaacaatcagctggtcgctggctacctgcaggagagcaacaccaagctgcaagagaagatcgacgtggtgcccatcgagcccgacgccctgttcctgggcctgcacggcagaaagctgtgcctggcctgcgtgaagtccggcgacgagatcagattccagctggaggccgtgaacatcaccgacctgagcaagaacaaggaggagaacaagcgcttcaccttcatcagaagcaacagcggccctaccacctccttcgagagcgccgcctgccccggctggttcctgtgcaccgcccaggaggccgacagacctgtctccctgaccaacaagcccaaagaatccttcatggtgaccaagttctacctgcaagaggatcagtga gcggccgcgggatccagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttagtcgactagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggctggggagagatcctagtttccatgctctaggagcatggctacgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggaattccagacgattgagcgtcaaaatgtaggtatttccatgagcgtttttcctgttgcaatggctggcggtaatattgttctggatattaccagcaaggccgatagtttgagttcttctactcaggcaagtgatgttattactaatcaaagaagtattgcgacaacggttaatttgcgtgatggacagactcttttactcggtggcctcactgattataaaaacacttctcaggattctggcgtaccgttcctgtctaaaatccctttaatcggcctcctgtttagctcccgctctgattctaacgaggaaagcacgttatacgtgctcgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttaaatatttgcttatacaatcttcctgtttttggggcttttctgattatcaaccggggtacatatgattgacatgctagttttacgattaccgttcatcgattctcttgtttgctccagactctcaggcaatgacctgatagcctttgtagagacctctcaaaaatagctaccctctccggcatgaatttatcagctagaacggttgaatatcatattgatggtgatttgactgtctccggcctttctcacccgtttgaatctttacctacacattactcaggcattgcatttaaaatatatgagggttctaaaaatttttatccttgcgttgaaataaaggcttctcccgcaaaagtattacagggtcataatgtttttggtacaaccgatttagctttatgctctgaggctttattgcttaattttgctaattctttgccttgcctgtatgatttattggatgttggaattcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagcag 2 Modified IL-1Ra insertatggagatcagacgcagaagcgtgcgccacctgatcagcctgctgctgttcctgctgtacagcgagaccgcctgccaccccctgggcaagaggccctgcaagatgcaggccttcagaatctgggacgtgaatcagaaaaccttctacatgcgcaacaatcagctggtcgctggctacctgcaggagagcaacaccaagctgcaagagaagatcgacgtggtgcccatcgagcccgacgccctgttcctgggcctgcacggcagaaagctgtgcctggcctgcgtgaagtccggcgacgagatcagattccagctggaggccgtgaacatcaccgacctgagcaagaacaaggaggagaacaagcgcttcaccttcatcagaagcaacagcggccctaccacctccttcgagagcgccgcctgccccggctggttcctgtgcaccgcccaggaggccgacagacctgtctccctgaccaacaagcccaaagaatccttcatggtgaccaagttctacctgcaagaggat cagtga 3Modified IL-1Ra insert atggagatcagacgcagaagcgtgcgccacctgatcagcctgctgctg(98% identical to SEQ ID ttcctgctgtat agcgagacagcctgccaccccctgggcaagaggccc NO: 2; bold letters aretgcaagatgcaggccttcagaatctgggacgtgaatcagaaaaccttcnucleotide substitutions tacatgcgcaacaatcagctggtcgctggctacctgcaggagagtaac within a codon (codon isaccaagctgcaagagaagatcgacgtggtgcccatcgagcccgacgcc underlined))ctgttcctgggcctgcacggcagaaagctgtgt ctggct tgcgtgaagtccggcgacgagatcagattccaa ctggaa gccgtgaacatcaccgac ctgagcaaaaacaaggaggagaacaagcgcttcaccttcatcagaagc aacagtggccctaccacctccttcgagagcgccgcctgccccggctggttcctgtgcaccgcccaggaggccgacagacctgtctccctgaccaacaagcccaaagaatccttcatggtgaccaagttctacctgcaagaggat caa tga 4Modified IL-1Ra insert atggagatcagacgcagaagcgtgcgccacctgatcagcctgctgctg(99% identical to SEQ ID ttcctgctgtat agcgagacagcctgccaccccctgggcaagaggccc NO: 2; bold letters aretgcaagatgcaggccttcagaatctgggacgtgaatcagaaaaccttcnucleotide substitutions tacatgcgcaacaatcagctggtcgctggctacctgcaggagagtaac within a codon (codon isaccaagctgcaagagaagatcgacgtggtgcccatcgagcccgacgcc underlined))ctgttcctgggcctgcacggcagaaagctgtgcctggcctgcgtgaagtccggcgacgagatcagattccagctggaggccgtgaacatcaccgacctgagcaagaacaaggaggagaacaagcgcttcaccttcatcagaagc aacagtggccctaccacctccttcgagagcgccgcctgccccggctggttcctgtgcaccgcccaggaggccgacagacctgtctccctgaccaacaagcccaaagaatccttcatggtgaccaagttctacctgcaagaggat caa tga 5Modified IL-1Ra insert atggagata agacgcagaagcgta cgccacctgatcagcctcctgctt (95% identical to SEQ ID ttcctgctgtat agcgagaca gcctgccatcccctgggcaagaggccc NO: 2; bold letters aretgcaagatgcaggccttcagaatctgggacgtgaatcagaaaaccttcnucleotide substitutions tacatgcgcaacaatcagctt gtcgctgga tacctgcaggagagtaac within a codon (codon is accaagctgcaagaaaagatcgacgtggtgcccatcgagcccgacgcc underlined)) ctcttcctgggcctgcacggcagaaagctgtgt ctggct tgcgtgaag tccggcgacgagatcagattccaactggaa gccgtgaacatcaccgac ctgagcaaa aacaaggaggagaacaagcgcttcaccttcatcaggagc aacagt ggccctaccacctca ttcgagagcgccgcctgt cccggctggttcctgtgcaccgcccaggaggccgacagaccc gtctccctgaccaat aagcccaaagaatcattcatggtgaccaagttctaccta caagaggat caa tga

In some embodiments, the IL-1Ra peptide encoded by the IL-1Ra insertcomprises IL-1Ra (see SEQ ID NO: 6, SEQ ID NO: 7 in Table 2 below).

TABLE 2 SEQ ID NO: DESCRIPTION SEQUENCE 6 IL-1Ra MEIRRRSVRH LISLLLFLFY(UNIPROT SETACHPLGK PCKMQAFRI O18999) WDVNQKTFYM RNNQLVAGYL QESNTKLQEK IDVVPIEPDA LFLGLHGRKL CLACVKSGDE IRFQLEAVNI TDLSKNKEENKRFTFIRSNS GPTTSFESAA CPGWFLCTAQ EADRPVSLTN KPKESFMVTK FYLQEDQ 7 IL-1RaMEIRRRSVRH LISLLLFLLY (encoded SETACHPLGK PCKMQAFRI by SEQWDVNQKTFYM RNNQLVAGYL ID NO: 2) QESNTKLQEK IDVVPIEPDALFLGLHGRKL CLACVKSGDE IRFQLEAVNI TDLSKNKEEN KRFTFIRSNS GPTTSFESAACPGWFLCTAQ EADRPVSLTN KPKESFMVTK FYLQEDQ

The transgene (e.g., nucleotide sequence encoding protein of interest)is operably linked to a promoter. In some embodiments, the promotercomprises the cytomegalovirus (CMV) promoter. The present invention isnot limited to the CMV promoter and may feature any appropriate promoteror portions of various promoters. Examples of promoters include CMVpromoter, hybrid CMV promoter, CAG promoter, human beta-actin promoter,hybrid beta-actin promoter, EF1 promoter, U1a promoter, U1b promoter, aTet-inducible promoter, a VP16-LexA promoter, chicken beta-actin (CBA)promoter, human elongation factor-1alpha promoter, simian virus 40(SV40) promoter, and herpes simplex virus thymidine kinase promoter. Insome embodiments, the promoter comprises a hybrid promoter. As anexample, Table 3 shows an IL-1 beta/IL-6 hybrid promoter (see also vande Loo et al., 2004, Gene Therapy 11:581-590). The present invention isalso not limited to the hybrid promoter shown in Table 3.

TABLE 3 SEQ ID NO: DESCRIPTION SEQUENCE 8 IL-1 beta/atccaagag ggagaagaag cccattggag IL-6 hybridatgatgccat aaaggaagtg gaagcgatat promotergataaaaatc atagtgccca ttcccaaata atcccagaag cagaagggaa aggagagaaatatccacaaa gacaggtgtg ggtacacaca acatttttca tactttaaga tcccagaggactcatggaaa tgatacaaga aaatgactca taagaacaaa tattaggaag ccagtgccaagaatgagatg ggaaattggg gaaaatgttg ggggcagatt gcttagttct gttctaagcaagagggtgaa caaggaagga acagctcact acaaagaaca gacatcactg catgtacacacaataatata agaactaacc catgattatt ttgcttgtct tcttgttcaa aatgattgaagaccaatgag atgagatcaa ccttgataac tggctggctt cggcatgatt agacacaagatggtatcagg gcacttgctg ctttgaataa tgtcagtctc ctgtcttgga agaatgacctgacagggtaa agaggaactt gcagctgaga aaggctttag tgactcaaga gctgaataattccccaaaag ctggagcatc ctggcatttc cagctcccca tctctgcttg ttccacttccttggggctac atcaccatct acatcatcat cactcttcca ctccctccct tagtgccaactatgtttata gcgagatatt ttctgctcat tggggatcgg aaggaagtgc tgtggcctgagcggtctcct tgggaagaca ggatctgata catacgttgc acaacctatt tgacataagaggtttcactt cctgagatgg atgggatggt agcagatttg ggtccaggtt acagggccaggatgagacat ggcagaactg tggagactgt tacgtcaggg ggcattgccc catggctccaaaatttccct cgagc ctctggccc caccctcacc ctccaacaaa gatttatcaaatgtgggatt ttcccatgag tctcaatatt agagtctcaa cccccaataa atataggactggagatgtct gaggctcatt ctgccctcga gcccaccggg aacgaaagag aagctctatctcccctccag gagcccagct atgaactcct tc

In some embodiments, the sc-rAAV vector is packaged within a capsid. Insome embodiments, the capsid comprises at least a portion of AAVserotype 1 (AAV1), AAV serotype 2, (AAV2), AAV serotype 3, (AAV3), AAVserotype 4, (AAV4), AAV serotype 5, (AAV5), AAV serotype 6, (AAV6),derivatives thereof, or combination thereof. For example, in someembodiments, the capsid comprises at least a portion of AAV serotype 2and at least a portion of AAV serotype 6, e.g., AAV2.5.

The composition, e.g., the composition comprising the sc-rAAV, may beintroduced into cells in a location of interest (e.g., in a horse). Forexample, in some embodiments when treating symptoms of osteoarthritis,the composition may be introduced into cells (e.g., chondrocytes,synoviocytes, e.g., type A, type B, etc.) in a joint via directintraarticular injection. In some embodiments, the composition isadministered to a joint, synovium, subsynovium, joint capsule, tendon,ligament, cartilage, or peri-articular muscle of the horse. The presentinvention is not limited to the aforementioned conditions (e.g.,osteoarthritis), the means of administration (e.g., intraarticularinjection), the location of interest (e.g., joint), or cell type (e.g.,chondrocytes, synoviocytes). For example, in some embodiments, othercell types that may be transduced may include mesenchymal stem cells.

The sc-rAAV transduces the vector into cells and the modified IL-1Rapeptide is expressed. In some embodiments, the IL-1Ra peptide isexpressed so as to provide the horse with a therapeutically effectiveamount of said modified IL-1Ra peptide effective for amelioratingsymptoms associated with various conditions such as osteoarthritis.

In some embodiments, introduction of the composition (e.g., the sc-rAAV)is performed once. In some embodiments, introduction of the composition(e.g., the sc-rAAV) is performed twice, e.g., a first time and a secondtime subsequent to the first time. In some embodiments, introduction ofthe composition is performed more than two times, e.g., three times,four times, five times, etc. The introduction of the composition asecond time may be performed at a time point after the time when themethod is first performed, e.g., after 3 months, 4 months, 5 months, 6months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, morethan one year, etc.

The composition may comprise any appropriate pharmaceutical composition.In some embodiments, the composition comprises a buffered solution. Insome embodiments, the buffered solution comprises phosphate bufferedsaline (PBS). In some embodiments, the composition further comprisessorbitol, e.g., 5% sorbitol. In some embodiments, the compositionfurther comprises a salt, e.g., NaCl. The concentration of salt may beany appropriate concentration, e.g., 350 mM NaCl, more than 350 mM NaCl,less then 350 mM, etc.

In some embodiments, the composition (e.g., the sc-rAAV) isco-administered with a secondary therapy. In some embodiments, thesecondary therapy comprises a therapeutic for OA or RA or any otherappropriate therapy for treating the symptoms of the condition.Non-limiting examples of secondary therapies for OA includeglucocorticoids, hyaluronan (viscosupplementation), platelet-richplasma, and recombinant, human IL-1Ra (Anakinra; Kineret®). For example,in some embodiments, the sc-rAAV is co-administered with glucocorticoidsor platelet-rich plasma.

The disclosures of the following U.S. Patents are incorporated in theirentirety by reference herein: US2008/0187576, US2009/0104155,KR2012041139, JP2015518816, WO2013151672, WO2008088895, U.S. Pat. Nos.8,529,885, 7,037,492, US20070128177, U.S. Pat. Nos. 6,491,907,8,999,948, US20150218586, U.S. Pat. No. 7,892,824, US20130295614,JP2002538770, JP2010516252, KR2002027450, KR2003028080, U.S. Pat. No.6,482,634, US20090105148, US20120232130, US20140234255, U.S. Pat. Nos.5,756,283, 6,083,716, WO2002038782, WO2007039699, WO2012047093,WO2014170470, WO2015018860, WO2015044292, WO2015158749, U.S. Pat. Nos.7,452,696, 6,943,153, 6,429,001, WO2015031392, WO2004092211.

Example 1

Example 1 describes administration of a sc-rAAV of the present invention(encoding IL-1Ra). The present invention is not limited to thedisclosure of Example 1.

Fifteen horses diagnosed with osteoarthritis in the knee are used for aclinical trial investigating administration of a sc-rAAV (encodingIL-1Ra) of the present invention. Twelve of the fifteen horses areadministered the sc-rAAV via intraarticular injection into the knee withosteoarthritis at 1×10¹⁰ viral genes per knee. The remaining threehorses are treated with a vehicle control. After three weeks, all horsesare evaluated for lameness. At the three-month time point, the threecontrol animals are given a second treatment of vehicle control. Threeof the 12 remaining previously treated horses are administered a secondadministration of the same sc-rAAV (via intraarticular injection intothe knee with osteoarthritis at 1×10¹⁰ viral genes per knee); three areadministered a second administration of a sc-rAAV (encoding IL-1RA) ofthe present invention that is different from the first sc-rAAV (viaintraarticular injection into the knee with osteoarthritis at 1×10¹⁰viral genes per knee); three are administered a second administration ofthe same sc-rAAV (via intraarticular injection into the knee withosteoarthritis at 1×10¹⁰ viral genes per knee) in combination with asecondary therapy (e.g., glucocorticoids and platelet-rich plasma); andthe remaining three are administered a second administration of thesc-rAAV (via intraarticular injection) into the knee with osteoarthritisat 1×10¹⁰ viral genes per knee) in combination with animmunosuppressant. The horses are evaluated for lameness at 3, 6, and 9weeks post-administration (of the second administrations).

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference cited in the presentapplication is incorporated herein by reference in its entirety.

Although there has been shown and described embodiments of the presentinvention, it will be readily apparent to those skilled in the art thatmodifications may be made thereto which do not exceed the scope of theappended claims. Reference numbers recited in the claims are exemplaryand for ease of review by the patent office only, and are not limitingin any way. In some embodiments, the figures presented in this patentapplication are drawn to scale, including the angles, ratios ofdimensions, etc. In some embodiments, the figures are representativeonly and the claims are not limited by the dimensions of the figures. Insome embodiments, descriptions of the inventions described herein usingthe phrase “comprising” includes embodiments that could be described as“consisting of”, and as such the written description requirement forclaiming one or more embodiments of the present invention using thephrase “consisting of” is met.

Any reference numbers recited in the below claims are solely for ease ofexamination of this patent application, and are exemplary, and are notintended in any way to limit the scope of the claims to the particularfeatures having the corresponding reference numbers in the drawings.

What is claimed is:
 1. A method of delivering a therapeuticallyeffective amount of interleukin-1 receptor agonist (IL-1Ra) peptide to ahorse in need thereof, said method comprising: introducing into alocation of interest a composition comprising a recombinantself-complementary adeno-associated virus (sc-rAAV), wherein saidsc-rAAV comprises: a. an engineered AAV capsid; and b. a vector packagedwithin the capsid, said vector comprising a modified IL-1Ra geneoperably linked to a promoter, the modified IL-1Ra gene is at least 95%identical SEQ ID NO: 2; wherein the sc-rAAV transduces the vector intocells in the location of interest, wherein the modified IL-1Ra gene isexpressed so as to provide the horse with the therapeutically effectiveamount of said IL-1Ra peptide.
 2. The method of claim 1, wherein saidhorse is diagnosed with or is at risk for developing osteoarthritis. 3.The method of claim 1, wherein the location of interest is a joint,synovium, subsynovium, joint capsule, tendon, ligament, cartilage, orperi-articular muscle of the horse.
 4. The method of claim 1, whereinthe composition is introduced into the location of interest via directintraarticular injection
 5. The method of claim 1, wherein the cells arechondrocytes, synoviocytes, or a combination thereof.
 6. The method ofclaim 1, wherein the method is performed a second time at a time pointafter a time when the method is performed first.
 7. The method of claim1, wherein the time point is at least 3 months.
 8. The method of claim1, wherein the method further comprises co-introducing a secondarytherapy to the location of interest in combination with the composition.9. The method of claim 8, wherein the secondary therapy comprises aglucocorticoid, hyaluronan, platelet-rich plasma, recombinant, horseIL-1Ra, or a combination thereof.
 10. The method of claim 1, wherein thepromoter comprises a CMV promoter.
 11. The method of claim 1, whereinthe engineered capsid comprises at least a portion of serotype AAV2 andat least a portion of serotype AAV6.
 12. The method of claim 1, whereinthe engineered capsid comprises at least a portion of serotype AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or acombination thereof.
 13. The method of claim 1, wherein the vectorfurther comprises SV40 and bovine growth hormone (bGH) polyadenylationsequences.
 14. The method of claim 13, wherein the vector furthercomprises SV40 splice donor (SD) and splice acceptor (SA) sites.
 15. Themethod of claim 1, wherein the vector comprises sc-rAAV2.5Hu-IL-1Ra. 16.A method of ameliorating symptoms of osteoarthritis in a horse, saidmethod comprising introducing into a location of interest a compositioncomprising a recombinant self-complementary adeno-associated virus(sc-rAAV), wherein said sc-rAAV comprises: a. an engineered AAV capsid;and b. a vector packaged within the capsid, said vector comprising amodified IL-1Ra gene operably linked to a promoter, the modified IL-1Ragene is at least 95% identical to SEQ ID NO: 2; wherein the sc-rAAVtransduces the vector into cells in the location of interest, whereinthe modified IL-1Ra gene is expressed so as to provide the horse with anamount of IL-1Ra peptide effective for ameliorating symptoms associatedwith osteoarthritis.
 17. The method of claim 16, wherein the location ofinterest is a joint, synovium, subsynovium, joint capsule, tendon,ligament, cartilage, or peri-articular muscle of the horse.
 18. Themethod of claim 16, wherein the composition is introduced into thelocation of interest via direct intraarticular injection
 19. The methodof claim 16, wherein the cells are chondrocytes, synoviocytes, or acombination thereof.
 20. The method of claim 16, wherein the method isperformed a second time at a time point after a time when the method isperformed first.
 21. The method of claim 16, wherein the time point isat least 3 months.
 22. The method of claim 16, wherein the methodfurther comprises co-introducing a secondary therapy to the location ofinterest in combination with the composition.
 23. The method of claim22, wherein the secondary therapy comprises a glucocorticoid,hyaluronan, platelet-rich plasma, recombinant, horse IL-1Ra, or acombination thereof.
 24. The method of claim 16, wherein the promotercomprises a CMV promoter.
 25. The method of claim 16, wherein theengineered capsid comprises at least a portion of serotype AAV2 and atleast a portion of serotype AAV6.
 26. The method of claim 16, whereinthe engineered capsid comprises at least a portion of serotype AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or acombination thereof.
 27. The method of claim 16, wherein the vectorfurther comprises SV40 and bovine growth hormone (bGH) polyadenylationsequences.
 28. The method of claim 27, wherein the vector furthercomprises SV40 splice donor (SD) and splice acceptor (SA) sites.
 29. Themethod of claim 16, wherein the vector comprises sc-rAAV2.5Hu-IL-1Ra.30. A method of delivering IL-1Ra peptide to a chondrocyte orsynoviocyte, said method comprising contacting the chondrocyte orsynoviocyte with a recombinant self-complementary adeno-associated virus(sc-rAAV) comprising: a. an engineered adeno-associated virus (AAV)capsid comprising at least a portion of serotype 2 and at least aportion of serotype 6; and b. a vector packaged within the capsid, saidvector comprising a modified IL-1Ra gene operably linked to a CMVpromoter, the modified IL-1Ra gene is at least 95% identical to SEQ IDNO: 2; wherein the sc-rAAV transduces the vector into the chondrocyte orsynoviocyte and the modified IL-1Ra gene is expressed to as to provideIL-1Ra peptide to the chondrocyte or synoviocyte.
 31. The method ofclaim 30, wherein the vector comprises sc-rAAV2.5Hu-IL-1Ra.
 32. Themethod of claim 30, wherein the vector further comprises SV40 and bovinegrowth hormone (bGH) polyadenylation sequences.
 33. The method of claim30, wherein the vector further comprises SV40 splice donor (SD) andsplice acceptor (SA) sites.
 34. A composition comprising a recombinantself-complementary adeno-associated virus (sc-rAAV), wherein saidsc-rAAV comprises: a. an engineered capsid comprising at least a portionof serotype 2 and at least a portion of serotype 6; and b. a vectorpackaged within the capsid, said vector comprises a nucleic acidsequence encoding a modified IL-1Ra peptide operably linked to a CMVpromoter, the nucleic acid sequence that encodes the modified IL-1Rapeptide is at least 90% identical to SEQ ID NO: 2;
 35. The compositionof claim 34, wherein the vector further comprises SV40 and bovine growthhormone (bGH) polyadenylation sequences.
 36. The composition of claim35, wherein the vector further comprises SV40 splice donor (SD) andsplice acceptor (SA) sites.
 37. The composition of claim 34, wherein thevector comprises sc-rAAV2.5Hu-IL-1Ra.
 38. A recombinantself-complementary adeno-associated virus (sc-rAAV) vector comprising amodified IL-1Ra gene operably linked to a CMV promoter, the modifiedIL-1Ra gene is at least 95% identical to SEQ ID NO:
 2. 39. The vector ofclaim 38 further comprising SV40 and bovine growth hormone (bGH)polyadenylation sequences.
 40. The vector of claim 39 further comprisingSV40 splice donor (SD) and splice acceptor (SA) sites.
 41. The vector ofclaim 38 comprising sc-rAAV2.5Hu-IL-1Ra.
 42. A method of repairingcartilage in a horse in need thereof, said method comprising:introducing into a location of cartilage a composition comprising arecombinant self-complementary adeno-associated virus (sc-rAAV), whereinsaid sc-rAAV comprises: a. an engineered AAV capsid; and b. a vectorpackaged within the capsid, said vector comprising a modified IL-1Ragene operably linked to a promoter, the modified IL-1Ra gene is at least95% identical SEQ ID NO: 2; wherein the sc-rAAV transduces the vectorinto cells in the location of cartilage, wherein the modified IL-1Ragene is expressed so as to provide the horse with IL-1Ra peptideeffective for repairing cartilage.
 43. The method of claim 42, whereinsaid horse is diagnosed with or is at risk for developingosteoarthritis.
 44. The method of claim 42, wherein the composition isintroduced into the location of cartilage via direct intraarticularinjection
 45. The method of claim 42, wherein the cells arechondrocytes, synoviocytes, or a combination thereof.
 46. The method ofclaim 42, wherein the method is performed a second time at a time pointafter a time when the method is performed first.
 47. The method of claim42, wherein the time point is at least 3 months.
 48. The method of claim42, wherein the method further comprises co-introducing a secondarytherapy to the location of cartilage in combination with thecomposition.
 49. The method of claim 48, wherein the secondary therapycomprises a glucocorticoid, hyaluronan, platelet-rich plasma,recombinant, horse IL-1Ra, or a combination thereof.
 50. The method ofclaim 42, wherein the promoter comprises a CMV promoter.
 51. The methodof claim 42, wherein the engineered capsid comprises at least a portionof serotype AAV2 and at least a portion of serotype AAV6.
 52. The methodof claim 42, wherein the engineered capsid comprises at least a portionof serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,AAV11, or a combination thereof.
 53. The method of claim 42, wherein thevector comprises sc-rAAV2.5Hu-IL-1Ra.
 54. A method of providinginterleukin-1 receptor agonist (IL-1Ra) peptide to an area ofinflammation, said method comprising: introducing into a location ofinflammation a composition comprising a recombinant self-complementaryadeno-associated virus (sc-rAAV), wherein said sc-rAAV comprises: a. anengineered AAV capsid; and b. a vector packaged within the capsid, saidvector comprising a modified IL-1Ra gene operably linked to a promoter,the modified IL-1Ra gene is at least 95% identical SEQ ID NO: 2; whereinthe sc-rAAV transduces the vector into cells in the location ofinflammation, wherein the modified IL-1Ra gene is expressed so as toprovide the cells in the location of inflammation a therapeuticallyeffective amount of IL-1Ra peptide effective for reducing inflammation.55. The method of claim 54, wherein the location of inflammation is ajoint, synovium, subsynovium, joint capsule, tendon, ligament,cartilage, or peri-articular muscle of the horse.
 56. The method ofclaim 54, wherein the composition is introduced into the location ofinflammation via direct intraarticular injection
 57. The method of claim54, wherein the cells are chondrocytes, synoviocytes, or a combinationthereof.
 58. The method of claim 54, wherein the promoter comprises aCMV promoter.
 59. The method of claim 54, wherein the engineered capsidcomprises at least a portion of serotype AAV2 and at least a portion ofserotype AAV6.
 60. The method of claim 54, wherein the engineered capsidcomprises at least a portion of serotype AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or a combination thereof.
 61. Themethod of claim 54, wherein the vector comprises sc-rAAV2.5Hu-IL-1Ra.62. A method of providing a horse in need thereof a therapeuticallyeffective amount of interleukin-1 receptor agonist (IL-1Ra), said methodcomprising: introducing into a location of interest a compositioncomprising a recombinant self-complementary adeno-associated virus(sc-rAAV), wherein said sc-rAAV comprises: a. an engineered AAV capsid;and b. a vector packaged within the capsid, said vector comprising amodified IL-1Ra gene operably linked to a promoter, the modified IL-1Ragene encodes IL-1Ra according to SEQ ID NO: 6 or SEQ ID NO: 7; whereinthe sc-rAAV transduces the vector into cells in the location ofinterest, wherein IL-1Ra is expressed so as to provide the horse withthe therapeutically effective amount of said IL-1Ra.
 63. A method ofameliorating symptoms of osteoarthritis in a horse, said methodcomprising introducing into a location of interest a compositioncomprising a recombinant self-complementary adeno-associated virus(sc-rAAV), wherein said sc-rAAV comprises: a. an engineered AAV capsid;and b. a vector packaged within the capsid, said vector comprising amodified IL-1Ra gene operably linked to a promoter, the modified IL-1Ragene encodes IL-1Ra according to SEQ ID NO: 6 or SEQ ID NO: 7; whereinthe sc-rAAV transduces the vector into cells in the location ofinterest, wherein IL-1Ra expressed so as to provide the horse with anamount of IL-1Ra effective for ameliorating symptoms associated withosteoarthritis.
 64. A method of delivering IL-1Ra peptide to achondrocyte or synoviocyte, said method comprising contacting thechondrocyte or synoviocyte with a recombinant self-complementaryadeno-associated virus (sc-rAAV) comprising: a. an engineeredadeno-associated virus (AAV) capsid comprising at least a portion ofserotype 2 and at least a portion of serotype 6; and b. a vectorpackaged within the capsid, said vector comprising a modified IL-1Ragene operably linked to a CMV promoter, the modified IL-1Ra encodesIL-1Ra according to SEQ ID NO: 6 or SEQ ID NO: 7; wherein the sc-rAAVtransduces the vector into the chondrocyte or synoviocyte and IL-1Ra isexpressed to as to provide IL-1Ra to the chondrocyte or synoviocyte. 65.A composition comprising a recombinant self-complementaryadeno-associated virus (sc-rAAV), wherein said sc-rAAV comprises: a. anengineered capsid comprising at least a portion of serotype 2 and atleast a portion of serotype 6; and b. a vector packaged within thecapsid, said vector comprises a nucleic acid sequence encoding amodified IL-1Ra peptide operably linked to a CMV promoter, the nucleicacid sequence encodes IL-1Ra according to SEQ ID NO: 6 or SEQ ID NO: 7.66. A method of repairing cartilage in a horse in need thereof, saidmethod comprising: introducing into a location of cartilage acomposition comprising a recombinant self-complementary adeno-associatedvirus (sc-rAAV), wherein said sc-rAAV comprises: a. an engineered AAVcapsid; and b. a vector packaged within the capsid, said vectorcomprising a modified IL-1Ra gene operably linked to a promoter, themodified IL-1Ra gene encodes IL-1Ra according to SEQ ID NO: 6 or SEQ IDNO: 7; wherein the sc-rAAV transduces the vector into cells in thelocation of cartilage, wherein IL-1Ra is expressed so as to provide thehorse with IL-1Ra effective for repairing cartilage.
 67. A method ofproviding interleukin-1 receptor agonist (IL-1Ra) peptide to an area ofinflammation, said method comprising: introducing into a location ofinflammation a composition comprising a recombinant self-complementaryadeno-associated virus (sc-rAAV), wherein said sc-rAAV comprises: a. anengineered AAV capsid; and b. a vector packaged within the capsid, saidvector comprising a modified IL-1Ra gene operably linked to a promoter,the modified IL-1Ra gene encodes IL-1Ra according to SEQ ID NO: 6 or SEQID NO: 7; wherein the sc-rAAV transduces the vector into cells in thelocation of inflammation, wherein IL-1Ra is expressed so as to providethe cells in the location of inflammation a therapeutically effectiveamount of IL-1Ra effective for reducing inflammation.